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Autocrine Stimulation by Estradiol-regulated Growth Factors of Rat Hormone-responsive Mammary Cancer: Interaction with the Polyamine Pathway

Departments of Medicine [A. M., C. W., P. F., L. B.] and Pathology, [L. D.] Division of Endocrinology, The Milton S. Hershey Medical Center, The Pennsylvania State University, Hershey, Pennsylvania 17033, and Unite d'Endocrinologie Cellulaire et Moleculaire, INSERM U148 Montpellier, France

We have recently shown that the integrity of the polyamine pathway is essential but not sufficient for expression of the mitogenic effect of estradiol (E₂) in the N-nitrosomethylurea-induced rat mammary tumor grown in vitro in the soft agar clonogenic assay. To elucidate the mechanism of E₂ action in this system, we tested whether E₂ may stimulate tumor growth through induction of secretory growth factor(s), as already proposed for human breast cancer cell lines in culture. Furthermore, we investigated the potential interaction between such "autocrine" control of tumor growth by E₂ and the polyamine pathway. Conditioned medium obtained from E₂-treated tumors (E₂-CM) but not from control tumors (C-CM) consistently stimulated colony formation when added to N-nitrosomethylurea-mammary tumors plated in soft agar under serum-free media conditions. Such growth-promoting effects of the E₂-CM was found to increase with increasing protein concentrations of the medium and was abolished by pretreatment of the medium with concanavalin A, heat, and trypsin. The addition of the polyamine biosynthetic inhibitor -difluoromethylornithine (1 mM) totally abolished the colony-stimulating effect of the E₂-CM. Exogenous administration of spermidine (0.1 mM) reversed the inhibitory effect of -difluoromethylornithine on colony formation and restored the action of the E₂-CM. Although the addition of polyamines alone did not affect the number of colonies formed, the administration of spermidine was found to significantly enhance in a dose-dependent fashion the colony-stimulating effect of suboptimal concentrations of E₂-CM. Attempts to identify the E₂-inducible growth factor in the E₂-CM and in N-nitrosomethylurea-mammary tumor specimens using monoclonal antibodies raised against the M_r 52,000 E₂-inducible protein gave negative results. We conclude that autocrine stimulation of tumor growth by E₂ is not limited to human breast cancer cell lines but also occurs in individual experimental mammary tumors grown in soft agar. Our results show that the polyamines must be present for the expression of this "autocrine" control of tumor growth by E₂. Finally, the identity of the E₂-induced growth factor operating in our system remains to be determined.

¹ To whom requests for reprints should be addressed, at Division of Endocrinology, The Milton S. Hershey Medical Center, P.O. Box 850, Hershey, PA 17033.

Received 8/ 9/85. Revised 10/15/85. Revised 11/26/85. Accepted 12/11/85.